Hi, everyone. I recently lysated cell culture with Trizol reagent, and I put my samples in -20C. But, some of my samples wont freeze, and I can see two separete phase appearing in them. I'm awaer, that thicker liquids wont freeze that good and because my cells are lysated they change the viscosity of the trizol. Even if they are still a liquid phase, they are still in -20C. Does it cause problems if they are still not frozen? Would the RNA will be degradated?
Trizol efficiently denatures and therefore inhibits RNase enzymes so there shouldn't be much degradation. But some RNA hydrolysis in the aqueous phase of Trizol may happen spontaneously. Although, this should be slow at -20C. Regardless, it should be fine for a day or so but its best not to leave the sample too long.
Ideally, its best not to stop the process at the trizol lysis stage. Either you can proceed with the extraction and store extracted RNA is in 1mM sodium citrate buffer, pH 6.4 frozen at -80°C. Or, you can store the source tissue in RNALater if you don't have time to extract.
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